Note: Descriptions are shown in the official language in which they were submitted.
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Fermentation process for the obtention - of a xanthane-type
polysaccharide and product to obtained
The present invention relates to a process for the
fermentation of carbohydrates for the preparation of a
05 polysaccharide of the xanthan type by means of microorganisms of
the Xanthomonas type, and to the polysaccharide obtained by this
process.
It is known that the aerobic fermentation of bacteria of
the Xanthomonas type in aqueous media with a pH of between 5.5 and
9, containing at least one source of carbon, a source of nitrogen,
- phosphate ions and trace elements, makes it possible to obtain a
polysaccharide composition of the xanthan type. The source of
carbon generally consists of carbohydrates, while the following
have been described as sources of nitrogen : dry distillery draff
in US patent 3.000.790, peptones in TAPPI 5, p. 442-445, by M.O.
BAGBY, I.A. WOLFF and M.C. CADMUS, yeast extracts in Biotechnol.
Bioeng. (1971) 13, p. 381, by R.A. MORAINE and P. ROGOVIN,
maceration liquors in US patent 3.335.447 or soya bean flours in
US patent 3.271.267 ; cereal flours, such as sorghum flours,
provide both the source of carbon and the source of nitrogen, as
described in US patent 3.271.267.
The products commonly used as sources of nitrogen
introduce insoluble impurities into the medium, which appear in
the polysaccharide isolated at the end of fermentation, this
implying, for certain applications of the polysaccharide,
treating the resulting product by various proc~sses, for example
filtration or flocculation, in order to remove these insoluble
impurities which lead to the formation of cloudiness in aqueous
polysaccharide solutions.
Furthermore, on the strength of its thickening property
which is virtually independent of temperature and p~, xanthan is
used in the assisted recovery of petroleum, as an edible additive
in pharmaceutical or cosmetic formulations, or in the preparation
of textiles or explosives, and it is of particular importance to
have a grade of xanthan having naturally a high thickening
.~
1312~0
property i.e. having aqueous solutions of high viscosity.
The present invention relates to a fermentation process
which, through the use of a suitably chosen source of nitrogen,
makes it possible to obtain a polysaccharide of xanthan type ~lith
05 a high thickening property, which contains few insoluble materials
and whose aqueous solutions are clear and only very slightly
colored, which is advantageous for numerous applications.
According to the invention, gelatin, and more particularly
a gelatin of low molecular weight, is used as the source of
nitrogen soluble in aqueous media. Two types of gelatin are known
to exist : type A gelatin, which is obtained by the acid
hydrolysis, for example with sulfuric acid, of the collagen
present in the skin or bones of pigs and cattle, and type B
gelatin, which is obtained by the basic hydrolysis of collagen,
especially with Ca(OH)2. The common gelatins, with a molecular
weight greater than or equal to 20000 are known sources of
nitrogen but they do not make it possible to achieve the
advantageous results of the invention. It is desirable to use
gelatins with an average molecular weight of less than 5000,
having practically no gelling strength ; the hydrolyzed gelatins
with a molecular weight between 1500 and 2500 are particularly
preferred.
It is possible to introduce into the fermentation medium 3
gelatin which has been hydrolyzed beforehand either by heating in
an aqueous acid medium at a pH <4 and at a temperature above
100C, or through the enzymatic action of a protease in the manner
described in French patent 1.501.821.
However, the gelatin can also be at least partially
hydrolyzed during the sterilization of the culture medium, before
the microorganisms are introduced, if the pH, temperature and
heating time are suitably chosen ; in this case, it is preferred
initially to introduce gelatins whose molecular weight, before
hydrolyzing sterilization, is no greater than 10.000.
When sterilization is carried out at an essentially
neutral pH or at a slightly acid pH, for very short times of less
a~ ~
than 5 minutes, the gelatin used must have a molecular weight of
less than 5000, preferably less than 3000.
The amount of gelatin introduced into the fermentation
medium is chosen so as to give a total nitrogen content of 0.1 g/l
û5 to 1 g/l.
The medium can also contain, in conventional manner, from
9/l to 50 9/l of carbohydrates such as glucose, native or
hydrolyzed starches, sucrose, levulose, fructose, maltose, sugar
beet molasses or sugar cane molasses, as well as 0.10 to 20 9/1,
preferably 0.5 to 5 9/l, of phosphates such as K2HP04, and one or
more trace elements, including magnesium at a concentration of
0.025 to 1 9/l of magnesium in the form of a soluble magnesium
salt such as magnesium sulfate, acetate, chloride or nitrate.
The bacteria which make it possible to obtain a xanthan by
fermentation in these media are generally of the genus Xanthomonas
and can belong to different species such as Xanthomonas begoniae,
Xanthomonas incanae, Xanthomonas vesicatoria, Xanthomonas
.
campestris and Xanthomonas phaseoli, and other species well known
to those skilled in the art ; strains of Xanthomonas campestris are
generally used.
Fermentation is carried out at a pH which can be between
5.5 and 9 and is preferably between 6.5 and 8. at a temperature
between 25C and 35C and preferably between 27C and 32C. The
medium is agitated and aerated in conventional manner ; under these
conditions, fermentation takes 1 to 6 days.
In a conventional manner, the process used to obtain the
polysaccharide according to the invention comprises several steps :
1/ Preparation of an inoculum from a strain of Xanthomonas
which was previously quick-frozen or lyophilized ;
2/ If appropriate, growing of the microorganisms in a
prefermentation medium ;
3/ Production of the polysaccharide in the fermentation
medium, which has been inoculated with the media obtained in step 1
or 2 and ;
4/ Isolation of the polysaccharide.
~3~2~
The polysaccharide is generally isolated by precipitation
through the introduction into the fermentation medium of a solvent
in which the polysaccharide is insoluble. Among the solvents
generally used, there may be mentioned lower alcohols such as
05 methanol, ethanol, isopropanol and butanol, or acetone; isopropanol
is preferred. Before the precipitation step, it is possible to heat
the reaction medium at a temperature between 80C and 130C for a
~ew minutes in order to destroy the bacteria.
A further possibility is to treat the medium before or
after sterilization, with an aliphatic dialdehyde such as glyoxal,
which is a known way of improving the dispersibility of
polysaccharides in water ; the xanthan is then precipitated in the
medium, isolated by filtration, washed with the precipitating
solvent and then dried and ground if necessary. In this case, 0.1
to 1 % by weight of glyoxal is used, relative to the weight of dry
xanthan ; in general, about 2 % is used. The xanthan can also be
treated with a dialdehyde after isolation, if this operation has
not already been carried out in the fermentation medium. An
advantageous process consists in wetting the xanthan powder with a
solution of glyoxal in aqueous isopropanol, prepared by mixing 5 to
10 ml of a 30 % aqueous solution of glyoxal with 150 ml of
isopropanol ; after a contact time of 20 to 40 minutes, the
solvents are removed by heating and the dry polysaccharide can then
be stored for subsequent use in its conventional applications.
To improve the fermentation conditions and, in particular,
to reduce the viscosity of the medium, fermentation on gelatin can
also be carried out in the presence of an oil and a surfactant, by
a process described in European patent application A-58364, which
uses a water-in-oil emulsion as fermentation medium or a process
described in European patent application A-187092, which uses an
oil-in-water emulsion as fermentation medium.
The invention further relates to the polysaccharide
composition of the xanthan type which is obtained by the
fermentation process according to the invention.
This polysaccharide composition, like all products of this
~ 3
. ~
type, consists of saccharide units of the mannose, glucose and
salified glucuronic acid type ; some of the hydroxyl groups are
esterified with acetic acid, and pyruvic acid is bonded via an
acetal group to some of the mannose units. The polysaccharide
05 composition according to the invention has a molecular weiyht of
more than 2,000,000.
Its aqueous solutions have a particularly high viscosity
which is greater than that of solutions of the xanthan produced by
fermentation in a medium containing soya bean flour or corn
maceration liquors as the source of nitrogen.
Thus an aqueous solu-tion containing 1% by
weight of KCl and 0.2% by weight of the polysaccharide
of the invention has a viscosity of at least 200 mPa.s,
measured on a Brookfield viscometer at 24C, whereas,
under the same conditions, that of an aqueous solution
of the polysaccharide obtained by fermentation on soya
bean flour is about 160 mPa.s.
The aqueous solutions of the invention polysaccharide have
a low optical density. For example, the solutions containing 0,2 %
by weight of polysaccharide have an optical density, measured at
620 nm, less than or equal to 0.2.
An Example of the way in which the invention is
carried out is now described below.
EXAMPLE
a) Preparation of the inoculum:
A revitalization culture medium MY consisting
of:
Glucose 10.0 g
Pentone 0.5 g
Yeast extract 0.3 g
Malt extract 3.0 g
Distilled water 1000 g
sterilized beforehand by heating for 20 min at 120C,
is inoculated with 3 ml of defrozen bacteria of the
Xanthomonas campestris strain deposited under no.
~ ~ 12 ~ 1J ~
NRRL.B 1459.
After inoculation, the mediuM is incubated at
30C for 12 to 18 hours, with agitation.
b) Prefermentation
05 The microorganisms of the inoculum are then
grown in a preculture fermentor containing 10 1 of a
medium consisting of:
Glucose 150.0 g
Gelatin 20.0 g
10 K2HP04 8.0 g
- MgS04 1.8 g
Antifoam 0 to 3.0 g
Waterqs for 10 1
The glucose is sterilized beforehand by heating
at pH 4, while the other constituents of the medium are
sterilized as a mixture, for example by heating at 120C
for 30 minutes at pH 4.5.
Prefermentation is continued for 20 hours before the
inoculum is used to inoculate the fermentation medium.
c) Fermentation and production of the polvsaccharide:
The fermentation medium for a fermenter with a
useful volume of 100 1 consists of:
Glucose 3 kg
Gelatin 200 g
K2HP04 80 g
MgS04 18 g
Antifoam0 to 30 g
Water~s for 100 1
The glucose is sterilized on its own at pH 4,
as previously, and the other constituents are sterilized
at 120C for 30 minutes at different pH values, leading
to gelatins with different molecular weights.
Before inoculation with the inoculum prepared
in step b, the pH of the medium is adjusted to 7 and
kept around this value throughout fermentation.
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,, .
In a conventional manner, the medium is agitated and
05 aerated throughout fermentation.
Table I indicates the results obtained using
two type A gelatins with a molecular weight of 5000, no
gelling strength and an isoelectric point of 6.3 or 6.6,
hydroly~ed during sterili~ation at different pH values.
Gelatin 1 is marketed by Rousselot under the
reference ASF and gelatin 2 under the reference HP50.
By way of comparison, a fermentation was also
carried out using soya bean flour as the source of
nitrogen.
The xanthan was precipitated at the end of the
fermentation process by introducing 200 1 of isopropanol
into the medium; it was then filtered off and dried by
heating in a strealD of air.
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